Original Article - Diabetes · and 3 months after bypass surgery. Immunomorphological changes of...

Original Article

Reduction of Macrophage Infiltration andChemoattractant Gene Expression Changes in WhiteAdipose Tissue of Morbidly Obese Subjects AfterSurgery-Induced Weight LossRaffaella Cancello,

1Corneliu Henegar,

1,2Nathalie Viguerie,

3Soraya Taleb,

1Christine Poitou,

1

Christine Rouault,1

Muriel Coupaye,1

Veronique Pelloux,1

Danielle Hugol,4

Jean-Luc Bouillot,5

Anne Bouloumie,3,6

Giorgio Barbatelli,7

Saverio Cinti,7

Per-Arne Svensson,8

Gregory S. Barsh,9

Jean-Daniel Zucker,1,10

Arnaud Basdevant,1

Dominique Langin,3

and Karine Clement1

In human obesity, the stroma vascular fraction (SVF) ofwhite adipose tissue (WAT) is enriched in macrophages.These cells may contribute to low-grade inflammation andto its metabolic complications. Little is known about theeffect of weight loss on macrophages and genes involved inmacrophage attraction. We examined subcutaneous WAT(scWAT) of 7 lean and 17 morbidly obese subjects beforeand 3 months after bypass surgery. Immunomorphologicalchanges of the number of scWAT-infiltrating macrophageswere evaluated, along with concomitant changes in ex-pression of SVF-overexpressed genes. The number of sc-WAT-infiltrating macrophages before surgery was higherin obese than in lean subjects (HAM56�/CD68�; 22.6 �4.3 vs. 1.4 � 0.6%, P < 0.001). Typical “crowns” ofmacrophages were observed around adipocytes. Drasticweight loss resulted in a significant decrease in macro-phage number (�11.63 � 2.3%, P < 0.001), and remainingmacrophages stained positive for the anti-inflammatoryprotein interleukin 10. Genes involved in macrophage

attraction (monocyte chemotactic protein [MCP]-1, plas-minogen activator urokinase receptor [PLAUR], and colo-ny-stimulating factor [CSF]-3) and hypoxia (hypoxia-inducible factor-1� [HIF-1�]), expression of whichincreases in obesity and decreases after surgery, werepredominantly expressed in the SVF. We show that im-provement of the inflammatory profile after weight loss isrelated to a reduced number of macrophages in scWAT.MCP-1, PLAUR, CSF-3, and HIF-1� may play roles in theattraction of macrophages in scWAT. Diabetes 54:2277–2286, 2005

Obesity is considered a chronic low-grade in-flammatory disease (1). The white adipose tis-sue (WAT) of obese subjects is characterizedby increased production and secretion of a

wide panel of inflammatory molecules, including tumornecrosis factor-� (2), interleukin (IL) 6 (3), transforminggrowth factor-� (4), monocyte chemotactic protein(MCP)-1 (5), and plasminogen activator inhibitor 1 (6).These inflammatory molecules may have local effects onWAT physiology in addition to their potential effects onother organs, if secreted (7–9). Macrophages infiltrate theWAT in both obese mice and humans (10,11). They maycontribute to the production of inflammatory moleculesand participate in the development of obesity-relatedcomorbidities. Resident macrophages were found in WATof obese subjects in three other studies (12–14). Macro-phage infiltration in WAT may be both a cause and aconsequence of the local and chronic inflammatory status inobesity (15). Leptin may stimulate diapedesis and infiltrationof macrophages into WAT (12). However, other yet unde-fined mechanisms may contribute to this phenomenon.

Weight loss improves inflammatory status in obesity andsubsequent comorbidities by decreasing numbers of circu-lating inflammatory molecules (1,16–18). We recentlyshowed that the expression of inflammation-relatedstroma vascular fraction (SVF) genes of WAT was de-creased by moderate weight loss (14). It is not knownwhether macrophage number and distribution are modi-

From 1Inserm “Avenir”, Paris 6 University EA3502 and Human ResearchCenter on Nutrition (CRNH), Hotel Dieu Hospital, AP/HP, Paris, France; 2SPIM“Sante publique et informatique medicale” laboratory, INSERM ERM202, Paris,France; 3INSERM UPS U586, Obesity Research Unit, Louis Bugnard Institute,Paul Sabatier University, Toulouse, France; the 4Department of Anatomo-Pathol-ogy, Hotel Dieu Hospital, Paris, France; the 5Department of Surgery, Hotel DieuHospital, Paris, France; the 6Cardiovascular Physiology Department, J.-W. GoetheUniversity, Frankfurt, Germany; the 7Institute of Normal Human Morphology-Anatomy, School of Medicine, Polytechnic University of Marche, Ancona, Italy;the 8Department of Internal Medicine, Research Centre for Endocrinology andMetabolism, Sahlgrenska Academy, Goteborg University, Goteborg, Sweden; the9Department of Pediatrics and Genetics, Howard Hugues Medical Institute,Beckman Center, Stanford University School of Medicine, Stanford, California;and the 10LIM & BIO, Paris-Nord University, Paris, France.

Address correspondence and reprint requests to Karine Clement, MD, PhD,Department of Nutrition, Hotel-Dieu Hospital, 1, Place du Parvis Notre-Dame,75004 Paris, France. E-mail: [email protected].

Received for publication 17 January 2005 and accepted in revised form 16May 2005.

CRP, C-reactive protein; CSF, colony-stimulating factor; GO, Gene OntologyConsortium; FDR, false discovery rate; HIF-1�, hypoxia-inducible factor-1�; IL,interleukin; MCP, monocyte chemotactic protein; ORO, orosomucoid; PLAUR,plasminogen activator urokinase receptor; RTqPCR, real-time quantitative PCR;SAA, serum amyloid A; SAM, significance analysis of microarrays; scWAT,subcutaneous WAT; SVF, stroma vascular fraction; WAT, white adipose tissue.

© 2005 by the American Diabetes Association.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked “advertisement” in accordance

with 18 U.S.C. Section 1734 solely to indicate this fact.

DIABETES, VOL. 54, AUGUST 2005 2277

fied together with changes in SVF gene expression duringsubstantial change of fat mass. We hypothesized thatweight loss is associated with major modifications ofinfiltrating macrophages in WAT and that candidate bi-omolecules, related to macrophage infiltration mecha-nisms, can be identified among the genes overexpressed inthe SVF. We systematically analyzed subcutaneous WAT(scWAT) samples of morbidly obese subjects before and 3months after surgery-induced weight loss. Here, we showthat fat reduction was associated with both a decrease innumber and modification of distribution of macrophagesin scWAT of obese subjects. After weight loss, remainingmacrophages stained positive for the anti-inflammatory mol-ecule IL10. The expression of chemoattractant genes (MCP-1,colony- stimulating factor [CSF]-3, and plasminogen activatorurokinase receptor [PLAUR]) was found in the SVF andincreased in obesity. Decreased expression of these geneswas observed after weight loss, suggesting a key role forthem in the macrophage recruitment to scWAT.

RESEARCH DESIGN AND METHODS

Subjects and WAT samples. Seventeen Caucasian morbidly obese womenundergoing laparoscopic Roux-en-Y bypass (19) and seven lean women (BMI21.95 � 0.83 kg/m2, aged 34 � 2.25 years, adipocyte mean size 62.4 � 2.28 �m)were enrolled at Hotel Dieu Hospital, Paris, France. Clinical and biologicaldata for obese subjects were recorded at their peak of weight and 3 monthsafter gastric bypass (Table 1). Fat mass was determined by biphotonicabsorptiometry (DXA; Hologic, Waltham, MA). ScWAT biopsies were per-formed by a surgeon after local anesthesia (1% xylocaine) of the periumbilicalarea. A portion of each scWAT biopsy was fixed for light and electron

microscopy, as described below, and immediately frozen for RNA extractionand analysis. To study the effects of weight loss on scWAT gene expression,we processed 10 high-quality RNAs from scWAT biopsies of the 17 collectedbefore/after bypass by microarray technique (Table 1). We examined differ-ences in gene expression between isolated SVFs and adipocytes from scWATspecimens of previously described overweight subjects (women, n � 9, BMI27.9 � 6.8 kg/m2) (14). The clinical investigations were performed accordingto the Declaration of Helsinki and approved by the ethics committees of HotelDieu (Paris, France). Signed informed consent was obtained from all subjects.Immunomorphological analysis of adipose tissue. ScWAT specimenswere fixed and processed as described (14). Immunohistochemical detectionof HAM56 (1:200; Dako Cytomation, Trappes, France), CD68 (1:100), and IL10(1:100) (Santa Cruz Biotechnologies, Heidelberg, Germany) were performedwith the avidin-biotin peroxidase method (20). Dewaxed sections (5 �m thick)were processed as previously described (14). Processed slide images wereacquired by a microscope-camera system (Nikon, Champigny sur Marne, France),and 400 cells were measured. Macrophage number was determined in all samplesby two independent observers. Four different randomly chosen areas within theprocessed slides labeled with HAM56 antibody were analyzed. Only HAM56�entire cells were counted, and the mean number of positive macrophages wasexpressed as percentages of mean adipocyte number.Transmission electron microscopy. Small scWAT fragments collected fromall obese patients before and after weight loss were fixed in 2% glutaraldehydein 0.1 mol/l phosphate buffer (pH 7.4), postfixed in 1% OsO4, dehydrated inethanol, and embedded an Epon-Araldite mixture (Epon; Multilab Supplies,Fetcham, U.K.) (Araldite; Fluka Chemie, Buchs, Switzerland). Thin sectionswere obtained with an MT-X ultratome (RMC, Tucson, AZ), stained with leadcitrate, and examined with a Philips CM10 transmission electron microscope(Royal Philips Electronics, Eindhoven, the Netherlands).Mature adipocytes and SVF cell separation. To compare adipocyte withSVF-overexpressed genes, we digested scWAT specimens of nine overweightfemale healthy patients (mean BMI 27.9 � 6.8 kg/m2), as previously described(14). Adipose tissue–derived macrophages included among SVF cells wereisolated as reported (12).

TABLE 1Clinical characteristics of 17 morbidly obese patients undergoing bypass

Bypass patients (n � 17)Mean (T0) Mean (3M) �Bypass microarrays (n � 10)

Sex Female — —Age (years) 43.5 � 4.10 (29–59) — —n � 10 41.5 � 4.0 (23–63) — —Height (m) 1.60 � 0.05 (1.52–1.79) — —n � 10 1.60 � 0.06 (1.59–1.65) — —Adipocyte size (�m) 98.7 � 1.2 (78.9–107.2) 83.3 � 1.3 (70.8–95) �15.4*n � 10 94.7 � 2.53 (77.76–107) 83.8 � 2.59 (66.03–94.69) �10.63†Weight (kg) 128.2 � 2.4 (92.3–164.4) 106.1 � 2.6 (80.0–143.0) �22.1*n � 10 123.1 � 1.6 (96.2–148.8) 104.5 � 1.7 (79.5–130.9) �18.53†Fat mass (kg) 65.15 � 3.5 49.65 � 2.80 �15.5†n � 10 67.33 � 4.03 51.38 � 3.54 �12.44†BMI (kg/m2) 48.0 � 6.8 (33.0–57.1) 39.7 � 6.8 (28.7–53.0) �8.3*n � 10 47.6 � 4.3 (43.0–57.0) 40.6 � 4.9 (34.0–49.0) �7.02†Glucose (mg/ml) 1.00 � 0.04 0.91 � 0.02 �0.09†n � 10 0.99 � 0.07 0.90 � 0.04 �0.09†Insulin (�U/ml) 14.2 � 2.2 12.8 � 3.1 �1.44†n � 10 12.8 � 3.5 7.2 � 1.1 �5.61†QUICKI 0.34 � 0.04 0.37 � 0.04 0.03†n � 10 0.33 � 0.02 0.37 � 0.02 0.04†Leptin (ng/ml) 49.7 � 6.7 30.9 � 5.6 �18.8†n � 10 51.9 � 3.4 31.0 � 4.1 �20.93†SAA (�g/ml) 35.03 � 9.5 15.86 � 3.1 �19.16†n � 10 35.15 � 10.13 12.62 � 3.5 �22.53†ORO (g/l) 0.94 � 0.03 0.90 � 0.04 �0.03‡n � 10 1.03 � 0.05 0.87 � 0.05 �0.16†CRP (�g/ml) 13.0 � 11.1 5.0 � 3.2 �8.0‡n � 10 30.0 � 9.6 5.6 � 1.1 �24.33‡

Data are means � SE (range). Metabolic and clinical parameters at baseline (T0) and 3 months after bypass (3M). � � strength of clinicalor biological changes before versus after surgery. Values in italics are clinical features of 10 morbidly obese subjects (of the 17 collected)whose scWAT was processed for microarray gene expression before/after surgery-induced weight loss. QUICKI, quantitative insulinsensitivity check index. *P 0.001, †P 0.01, ‡P 0.05.

MACROPHAGE INFILTRATION IN ADIPOSE TISSUE

2278 DIABETES, VOL. 54, AUGUST 2005

Total RNA extraction, mRNA amplification, and microarrays. Total RNAfrom scWAT was prepared using the RNeasy RNA Mini Kit (Qiagen, Courtaboeuf,France). One microgram of total RNA from each sample preparation wasamplified using the MessageAmp RNA kit (Ambion, Austin, TX), and 3 �g aRNAwas Cy-dye labeled using the CyScribe first-strand cDNA labeling kit (AmershamBiosciences, Orsay, France) (21 [available at http://cmgm.stanford.edu/pbrown/protocols/index]). aRNA extracted from each scWAT sample before surgery waslabeled with Cy3 dye, while the aRNA from each scWAT sample obtained 3months after surgery was labeled with Cy5 dye. The hybridization, washing, andscanning procedures were performed as described (14). Several quality cross-checks (for total RNA quality, aRNA quality, dye incorporation efficiency, etc.)and microarray “dye swap” experiments were performed.Statistical analysis of microarray data. Only spots with a regressioncoefficient 0.6 and a signal intensity 50% above background in both Cy5 andCy3 channels were retrieved from the Stanford Microarray Database (available athttp://genome-www5.stanford.edu/). Genes with significant changes in expres-sion were identified using the significance analysis of microarrays (SAM) proce-dure (available at http://www-stat.stanford.edu/�tibs/SAM/) (22). We used aselection threshold yielding the lowest median estimate of 0.05 false-positivegenes (false discovery rate [FDR] of 5%). We also checked that all significantgenes were sorted by SAM with 10% FDR when comparing the profiles of geneexpression before and after weight loss in obese subjects. Hierarchical clustering

of the significant genes was computed for the ratios after normalization. Theclusters were visualized by Tree-View (23).Functional annotation of significant genes in adipocytes and the

SVF. Functional profiling of differentially regulated genes in adipocytes andthe SVF was performed based on the Gene Ontology Consortium (GO[available at http://www.geneontology.org]) terms (24). Using an “in-house”automated discriminatory annotation procedure, we identified GO categoriesthat annotate genes differentially expressed in adipocytes and the SVF. Thedetailed procedure of this method, implemented into an algorithm calledFunCluster (C. Hengar et al., unpublished data), is available online assupplementary data (available at http://corneliu.henegar.info/FunClus-ter.htm). We clustered together all related GO categories, based on sharing ofa significant number of annotated genes. The gene annotation procedure wasperformed separately for each of the three available GO ontologies: molecularfunction, biological process, and cellular component. The biological processannotations presented in this article were the more exhaustive and immedi-ately comprehensive ones. Molecular function–, cellular component–, andKEGG (Kyoto Encyclopedia of Genes and Genomes)-based annotations areavailable as supplementary online data. The P value of gene enrichmentsignificance was calculated for each of the identified clusters by using aunilateral Fisher’s exact test adjusted for multiple testing errors with the

FIG. 1. Macrophage infiltration of scWAT in obese subjects before (T0) and 3 months after (3M) weight loss surgery. A–D: The morphology ofscWAT of one representative morbidly obese study subject (BMI 50 kg/m2) is shown. Macrophage infiltration was examined first by routineMayer’s hematoxylin and eosin staining (A; �100). Infiltrating macrophages formed a typical crown. Immunopositivity for HAM56 (B; �100) andCD68 (C; �100). D: Lack of immunostaining for IL10 (�100). Adipocytes tested negative to all these markers. E–H: Immunomorphology of thescWAT sample obtained 3 months after surgery. Routine Mayer’s hematoxylin and eosin staining (E; �100): absence/disorganization ofmacrophage crowns. Immunopositivity for HAM56 (F; �100) and CD68 (G; �100). H: Immunostaining in resting macrophages for IL10 (�100).Percentages of macrophages were estimated in scWAT specimens before/after weight loss and in lean subjects, ***P < 0.001 (K). Individualdecreases in percentage of macrophages in 17 morbidly obese subjects 3 months after weight loss surgery (***P < 0.001) (K).

R. CANCELLO AND ASSOCIATES


Benjamini and Hochberg FDR correction approach. The clusters were rankedbased on the statistical significance of the gene space coverage.Genes predominantly expressed in human monocyte–derived macro-

phages. The SVF gene list was crossed with results for human macrophagepredominantly expressed gene selection to estimate the contribution ofmacrophage gene expression in adipose tissue–derived SVF cells. Humanmacrophages from 15 healthy nonobese donors were differentiated andcultured as described (25). Total cellular RNA was isolated from differentiatedmacrophages using the RNeasy kit (Qiagen, Hilden, Germany). Target prepa-ration was performed according to the manufacturer’s instructions (Af-fymetrix, Santa Clara, CA). Aliquots of the target preparation (15 �g copyRNA) were hybridized to individual U133A-DNA microarrays and thenwashed, stained, and scanned (GeneArray scanner G2500A; Hewlett Packard)according to procedures developed by the manufacturer (Affymetrix).Scanned output files were visually inspected for hybridization artifacts andthen analyzed with MAS-5 software (Affymetrix). Sixty-eight nonmacrophageU133A expression profiles were obtained from the Symatlas database (avail-able at http://symatlas.gnf.org/SymAtlas/) (26). Cancer tissues, fetal tissues,and cell lines present in the Symatlas database were excluded from analysis.The signal of each gene was normalized to the mean signal intensity of eachDNA microarray (27). The average signal for each gene was calculated fromthe 15 macrophage and the 68 duplicate nonmacrophage expressionprofiles. The level of macrophage expression of each gene analyzed wasranked, with genes having maximal expression in macrophages ranked 1and genes having minimal expression in macrophages ranked 69, aspreviously detailed (28).Real-time quantitative PCR. We validated the changes in gene expression onRNA from whole scWAT, isolated adipocytes, the SVF, and macrophages byreverse transcription and real-time quantitative PCR (RTqPCR) (21). 18S ribo-somal RNA amplification (Ribosomal RNA Control TaqMan assay kit; AppliedBiosystems, Foster City, CA) was used as normalization control. The primers andTaqMan probes for mRNA were obtained from Applied Biosystems.Conventional statistical analysis. General statistical analysis was per-formed with JMP statistics software (SAS Institute, Cary, NC). For genesanalyzed by RTqPCR, significant differences were determined by Wilcoxonnonparametric paired test (before/after surgery; adipocyte vs. SVF) andStudent’s t test with unequal variance (obese vs. lean subjects). Correlations

were examined by the nonparametric Spearman’s rank correlation test. P 0.05 was the threshold for significance.Online supplementary data. Materials and methods details and annexes tofigures are available online at http://corneliu.henegar.info/FunCluster.htm.

RESULTS

Macrophage infiltration in scWAT before and after

weight loss. The morphology of scWAT was examined bylight microscopy before (T0) and 3 months after (3M)bypass. Macrophage infiltration was observed in scWAT ofall obese subjects (Fig. 1A, T0). The morphology ofmacrophages in scWAT parenchyma differed markedlybetween obese and lean subjects. In lean individuals,single isolated macrophages surrounded endothelial trees,sometimes reaching small capillaries between adipocytes(annex to Fig. 1, see ONLINE SUPPLEMENTARY DATA). In obesesubjects, macrophages spread into the parenchyma ofscWAT (Fig. 1A–D). In some areas, macrophages sur-rounded mature adipocyte cytoplasm in typical “crowns”(Fig. 1A–D). They exhibited triglyceride molecules in theircytoplasm and resembled foam cells (Fig. 1A–D). Trans-mission electron microscopy analysis confirmed the pres-ence of macrophages very close to adipose cellmembranes. Macrophage projections were directed toregions of adipocyte cytoplasm very rich in lipofuscininclusions, typical of injured cells (annex to Fig. 1). Thepercentage of macrophages was significantly higher inscWAT of obese patients than lean subjects: 22.6 � 4.3%(95% CI 16.8–28.3) vs. 1.4 � 0.6% (0.95–1.8) in obese vs.lean subjects (P 0.001) (Fig. 1I). All macrophages testedpositive for HAM56, a marker of mature macrophages (Fig.1I). Macrophages stained for CD68, a marker of the phago-cytic activity (Fig. 1C). Before surgery, no staining was foundfor the anti-inflammatory cytokine IL10 (Fig. 1D).

Significant correlations were found between the numberof macrophages and both BMI (� � 0.48, P � 0.0009) andmean adipocyte size (� � 0.53, P � 0.0008). A significantpositive correlation was found between macrophage num-ber and patient age (� � 0.677, P 0.05). The associationsbetween BMI and adipocyte size and macrophage numberremained significant after age adjustment.

A significant decrease in plasma concentrations of lep-tin, insulin, and C-reactive peptide (CRP), as well as twoacute-phase proteins serum amyloid A (SAA) and oroso-mucoid (ORO), were observed after bypass (Table 1).Three months after weight loss, a significant reduction ofinfiltrating macrophages (�11.63 � 2.3% [95% CI �8.43 to�14.8], P � 0.01) (Fig. 1I–K) was observed. The tissuelocalization of the remaining macrophages was substan-tially different: macrophages were near blood vessels, andcrowns had disappeared (Fig. 1 3M, E–H). The remainingmacrophages still stained for HAM56 and CD68 (Fig.1F–G). To test the hypothesis of phenotypic change fromproinflammatory (M1) to anti-inflammatory (M2) macro-phages after rapid weight loss, we examined the stainingof scWAT-infiltrating macrophages for the most potentanti-inflammatory cytokine, IL10. Staining was almost ab-sent in T0 macrophages but was strong in 3M remainingmacrophages (Fig. 1H). Change in macrophage numberwas significantly correlated with changes in plasma levelsof two acute-phase proteins, SAA (� � 0.71, P 0.05) andORO (� � 0.69, P 0.05). A borderline significant corre-lation was observed for change in CRP variation (� � 0.58,

TABLE 2Comparison between microarrays and RTqPCR ratios of SVF-and adipocyte-overexpressed genes

GeneRatio

microarrayQ value

5%Adipocyte-to-SVFratio (RTqPCR)

SVFIL1Ra 0.24 0.014 0.41*MCP-1 0.36 0.014 0.28*TNF� 0.23 0.014 0.10*A2M 0.38 0.014 0.49*CD68 0.14 0.014 0.13*ITGAL 0.84 0.37 0.11*CSFR1 0.54 0.014 0.09*MMP9 0.27 0.014 0.08*SDF1/CXCL12 0.62 0.014 0.19*HF1 0.54 0.73 0.20*SERPA3 0.81 0.088 0.21*MIP-3� 0.76 0.091 0.30*

AdipocyteFGF13 2.17 0.014 6.10*PTGER3 3.56 0.014 4.70*CES1 4.06 0.014 11.7*

The following genes overexpressed in the SVF were validated byRTqPCR: IL1Ra, MCP-1, TNF�, A2M, CD68, ITGAL, CSF1R, MMP9,SDF1/CXCL12, HF1, SERPA3, MIP-3�. FGF13, PTGER3, and CES1,genes were preferentially expressed in isolated mature adipocytes,confirming the microarray data ratios. Q values are for differences ingene expression between adipocytes and SVF after application ofSAM procedure to microarray ratios. Adjustment was performed fortesting multiple hypotheses. Corresponding adipocyte/SVF meanratios obtained by RTqPCR are given. Normalization was performedwith 18S mRNA for RTqPCR experiments. *P 0.05, Wilcoxonnonparametric paired test.



P � 0.09). No significant correlation was observed be-tween macrophage number and clinical and metabolicparameters, neither with fasting insulin levels and quanti-tative insulin sensitivity check index nor with changes ininsulin sensitivity after weight loss (annex to Table 1).Determination of SVF-overexpressed genes by mi-

croarray analysis. Since macrophages enriched the SVFof scWAT of obese subjects and their number decreasedby weight loss, we examined the expression profile of SVFgenes to determine whether some of them may be involved inthe mechanisms of macrophage recruitment to scWAT. Toselect SVF-overexpressed genes, we used results of a previ-ously established gene expression profiling experiment per-formed after dissociation of adipocytes and the SVF (14). Weextracted the differentially expressed genes using the SAMprocedure and then searched for enriched functions that bestcharacterized the sets of adipocyte- and SVF-related genes.With a 5% FDR, SAM isolated 10,432 cDNAs, among which6,587 exhibited increased expression in adipocytes (ratiorange 1.04–16.31), while 3,845 were overexpressed in theSVF (ratio range 1.06–8.95). GO biological process annota-tions were found for 3,098 genes (with Locuslink ID), with1,743 and 1,355 overexpressed in adipocytes and the SVF,respectively (online supplementary data). We validated ex-pression changes for 15 randomly chosen genes, 12 withincreased expression in the SVF (P 0.05), and 3 mainlyexpressed in mature adipocytes (P 0.05) (Table 2). To findthe set of genes and functions that best characterized theSVF, we used our in-house approach based on GO “biologicalprocess” terms. Forty significant functional clusters werefound for adipocyte-overexpressed genes, while 30 signifi-cant clusters characterized SVF-overexpressed genes (onlinesupplementary data).

Figure 2 shows the first GO-annotated clusters rankedby statistical significance. The enriched functions that bestcharacterized the genes overexpressed in adipocytes rep-resent their well-known metabolic and secretory proper-ties (Fig. 2A). In contrast, the most significant functionscharacterizing the genes overexpressed in the SVF wererelated to inflammatory or immune processes (Fig. 2B).The GO terms that grouped genes in the first five signifi-cant clusters were “defense response,” “chemotaxis,” “hu-moral immune response,” “antigen processing,” and“inflammatory response” (Fig. 2B). Other significant func-tional clusters characterizing the SVF were related to“regulation of cell proliferation,” “regulation of transcrip-

tion,” “protein biosynthesis,” and “extracellular matrixorganization and biogenesis” GO terms (P 0.001–0.05).

We performed a set of RTqPCRs to validate the expres-sion levels of seven SVF inflammatory genes. As shown inTable 3, all selected genes (MCP-1, �-2–macroglobulin,tumor necrosis factor-�, CD68, IL10, IL1 receptor antago-nist, and hypoxia-inducible factor-1� [HIF-1�]) were sig-nificantly overexpressed in scWAT-derived macrophagescompared with isolated adipocytes.SVF genes modulated by weight loss. We subsequentlyfocused on the set of 260 inflammatory genes of SVF cellsto study the modulation of expression of these genes afterweight loss and to examine if candidate genes of macro-phage chemoattraction might be present among them. Wecompared the list of these genes with the genes differen-tially expressed in scWAT after 3 months’ weight loss andrecovered 196 significant common genes. Among them,119 (46%) exhibited a change in expression with 10% FDR,and 90 (35%) cDNAs exhibited differential expression with5% FDR. As shown in Fig. 3, after clustering, 49 and 41gene transcripts were under- and overexpressed, respec-tively, after weight loss. The set of upregulated genesincluded mostly those for complement-related factors andthe major histocompatibility complex. The cluster ofdownregulated genes was composed of genes annotatedby the following terms: “defense and immune response”(IL6 and IL6R), “chemotactism” (such as CCL1, MCP-1,and CCL6), and “positive regulation of cell proliferation”or “cell-cell signaling” (CSF-3 and regulator of macrophagedevelopment and survival). Two clones of the � subunit ofthe HIF-1� gene printed on the array were found in thiscluster of downregulated genes (Fig. 3). To estimate thecontribution of macrophage gene expression in the SVF, weused expression profiles from cultured human macrophagesand 68 other tissues and cell types and the ranking proceduredescribed in RESEARCH DESIGN AND METHODS. The list of 90inflammatory SVF-related genes was crossed with a list ofgenes predominantly expressed in human macrophages thathas been previously reported (28): 39 of 90 SVF-related genes(43.3%) were macrophage-overexpressed and 33 genes(36.6%) downregulated. Ten of the 90 regulated genes wereranked as number one for macrophages, showing that mac-rophages are the major site of expression of these genes andstrongly contribute to their expression in the SVF (Table 4,online supplementary data). Three of these genes havepreviously been found to be predominantly expressed inmacrophages (28). For 27 additional genes, macrophageexpression was ranked in the top 15, suggesting that macro-phage expression of these genes contributes to their expres-sion in the SVF (Table 4, online supplementary data). For the34 genes with the lowest ranking (ranks 36–69), macrophageexpression is probably limited or expression of them is dueto other cell types in the SVF.Genes involved in macrophage recruitment and hyp-

oxia. Among the inflammatory genes modulated by weightloss (Fig. 3), four genes known to be involved in macro-phage tissue attraction and regulation clustered together:MCP-1, CSF-3, and PLAUR. These genes play roles inchemotaxis and maintenance of circulating monocytes intissue, developing an inflammatory state (29–31). Thehypoxia-inducible gene HIF-1� was also located in thiscluster. As shown in Fig. 4, levels of expression of MCP-1,

TABLE 3Comparison of gene expression in isolated adipocytes and adi-pose tissue-derived macrophages by RTqPCR

Gene Adipocytes Macrophages

MCP1 0.047 � 0.011 0.28 � 0.1**A2M 0.016 � 0.003 0.204 � 0.1**TNF� 0.024 � 0.006 0.356 � 0.062***CD68 0.027 � 0.005 1.55 � 0.24**IL10 0.001 � 0.0002 0.102 � 0.038***IL1Ra 0.005 � 0.0036 1.06 � 0.3***HIF-1� 0.037 � 0.0025 0.549 � 0.053***

Data are mean RTqPCR ratio � SE. Macrophages were isolated fromadipose tissue SVF cells and gene expressions of MCP-1, A2M, TNF�,CD68, IL10, IL1Ra, and HIF-1� subunit were compared with those ofisolated mature adipocytes. mRNA arbitrary units were normalizedusing 18S ribosomal RNA.



CSF-3, HIF-1�, and PLAUR genes were significantly higherin scWAT of obese than in that of lean women (P 0.05).After weight loss, we confirmed the significant decrease inexpression of all these genes (Fig. 4). In the RTqPCRvalidation procedure, IL10 and IL1 receptor agonist genes,two anti-inflammatory cytokines, and the regulatory cyto-kine gene IL12A were upregulated after surgery-inducedweight loss (annex to Fig. 4).

DISCUSSION

Macrophage infiltration of scWAT may participate in thelow-grade inflammation associated with human obesity(10,11). We demonstrate that weight loss is associatedwith a reduction in the number and modified distribu-tion of macrophages infiltrating scWAT of obese sub-jects. We confirmed that the increase in macrophageinfiltration is significantly correlated with BMI andadipocyte size (10,12,13). Nevertheless, we better char-acterized specific histological features of infiltrating

macrophages in WAT. In lean women, the rarely observedmacrophages were dispersed among mature adipocytes andlocated near blood vessels. They probably correspond to thenumber of resident macrophages in scWAT that may even-tually respond to infections. In scWAT of obese subjects,macrophages formed typical crowns, completely surround-ing single adipocytes. They contained triglyceride particles incytoplasm and resembled foam cells or phagocytes (32–35).This feature was never observed in lean subjects. On trans-mission electron microscopy, macrophage projections fre-quently held adipocyte membranes that contained cytoplasmlipofuscin inclusions, a typical feature of stressed, aged, anduseless cells (36,37). We found no evidence of apoptosis.Altogether, morphology, distribution, and the staining pat-tern observed suggest a phagocytic activity of macrophagesdirected toward adipocytes in human scWAT. The potentialcapacity of macrophages to phagocyte adipose cells and themechanisms involved warrant evaluation.

After weight loss (3 months), we observed a reduced

FIG. 2. Clustering of functions of genes overexpressed in isolated human adipocytes and the SVF. Clusters of enriched functions are listed by P

value. The four most significant annotated gene clusters for adipocytes (A; red bars) and the five most significant ones for the SVF (B; green bars)are shown. The gene number annotated by a given GO term (Biological Process branch) is indicated in parenthesis. The reported P value is givenby Fisher’s exact test corrected by FDR.



number of macrophage cells and a disappearance ofcrowns. Despite substantial amelioration of fasting insulinand insulin sensitivity after surgery, assessed by quantita-tive insulin sensitivity check index, we failed to detect asignificant correlation between these parameters and thenumber of scWAT-infiltrating macrophages. The pairedcorrelation study performed had a weak statistic powerdue to the sample size (n � 17), and we did not detectsignificant correlations for insulin sensitivity or fastinginsulin levels in these extremely obese subjects. Thenumber of scWAT macrophages remained elevated 3months after bypass, despite a general trend toward adecrease in the entire macrophage population. Reductionin the number of macrophages in scWAT may neverthelessdecrease obesity comorbidities via improvement of low-grade inflammation. The correlations between reductionof macrophage number and decrease in two major acute-phase protein levels (ORO and SAA) suggest the existenceof a relationship or of a cross-talk between scWAT-infiltrating macrophages and the systemic inflammatorystate sustained by mature adipocytes (8). A larger cohort isneeded for the borderline significant correlation observedfor CRP variations. Noteworthy, the microarray subgrouphad a higher CRP level than the whole bypass group. Asdescribed above, they were chosen for the good rate ofRNA extraction and quality from scWAT specimens, and a

higher CRP level was observed by chance. RTqPCR vali-dations performed on scWAT samples from patients withvarious CRP levels were homogeneous, suggesting thatCRP levels did not influence gene expression.

The important role of the SVF in immunity and inflam-matory processes (38) was confirmed by our automatedprocedure of annotation. At least 30–50% of SVF inflam-matory genes were highly sensitive to weight loss. Thisobservation agrees with our previous results in less obesesubjects after a 4-week very-low-calorie diet (14). Weshow here that decreased expression of inflammatorymarkers is associated with decreased macrophage numberand increased expression of molecules with anti-inflam-matory properties such as IL10 (39) by resting macro-phages. These findings strongly support the existence of aphenotypic M1/M2-like shift of macrophages and provideadditional clues to improvement of the inflammatory sta-tus of scWAT by modification of macrophage number andphenotype. Many of the SVF-overexpressed genes andtheir functions correspond to recognized properties ofmacrophages. Of the 90 SVF-enriched inflammatory genes,at least 39% were expressed at high levels in macrophagescompared with other tissues and cell types (28). Enrich-ment of macrophages in the SVF is probably the principalreason for the inflammatory state observed in scWAT ofmorbidly obese subjects. The contribution of a small

FIG. 2—Continued.



number of other immune/nonimmune cells to the SVFgene expression profile cannot be excluded. Preadipo-cytes exhibit close similarities in gene expression tomacrophages and may convert to macrophages in inflam-matory microenvironments (40–42).

The mechanisms responsible for macrophage recruit-ment and activation in human hypertrophic scWAT areunknown. Macrophages derived from bone marrow pre-cursors and circulating monocytes may infiltrate scWAT(10,12). Paracrine, autocrine, and endocrine signals as wellas mechanical modifications (i.e., hypertrophy/hyperpla-sia) of WAT cells may also play roles in such mechanisms.

Several chemokines, such as MCP-1 and CSF-3, but inaddition other hitherto undefined factors, may be involvedin macrophage recruitment into scWAT (5,31,43,44). Somechemotactic factors (and/or their receptors) exhibiteddecreased expression after weight loss and clusteredtogether (Figs. 3 and 4). Levels of expression of chemo-tactic molecules (MCP1, CSF3, and PLAUR genes) inscWAT of morbidly obese subjects were significantlyhigher than in that of lean subjects. The overexpression ofHIF-1� in morbid obesity and the decrease in its expres-sion after weight loss also indicate the contribution oflocal scWAT hypoxia to macrophage movement, as sug-

FIG. 3. Clusters of SVF genes with expression significantly up- or downregulated by weight loss. Each row represents a single gene. Each columnrepresents the individual ratio of each gene for one obese subject after/before weight loss surgery. BMI (kg/m2) before (T0) and three monthsafter (3M) weight loss. For each gene, the log2 ratio intensity after/before weight loss of 10 obese subjects is shown by colored blocks. Green,red, and black lines represent log2 ratio intensity of each gene transcripts below, above, and equal to the median of array intensity, respectively,after the normalization procedure. Gray blocks are missing signals. The dendrogram shows that in the scWAT of all obese subjects, expressionof some inflammatory genes was upregulated (red), while that of others was downregulated (green) after weight loss. Genes shown in bold arecandidates for their involvement in macrophage chemoattraction and hypoxia.



gested (8). Tissue and cellular hypoxia is a well-knowncause of macrophage attraction and retention, particularlyin tumors and atherosclerotic plaque (30). The hypoxicstate is relatively common in obese patients (8,45). Amongthe clusters of genes downregulated by weight loss, wefound, together with HIF-1�, several genes known to betargets of this transcription factor (such as IL6, superoxide dismutase 2, C-X-C chemokines, and PLAUR)(46,47), suggesting that “codownregulation” of these mol-ecules occurs during weight loss. Progression of adipositycould, together with worsening of local microhypoxia andpersistent stimulation of chemotactic cytokines, enablemacrophage infiltration and maintenance. Such a role wassuggested for MCP-1 and CSFs (48,49). This hypothesiswarrants further evaluation, in particular the analysis ofthe modification of these biomolecules at the protein level.

In conclusion, we show that the improvement of theinflammatory profile of morbidly obese patients afterweight loss is associated with reduced number of infiltrat-ing inflammatory cells in scWAT and transcriptional mod-ulation of a cocktail of immune and inflammatorybiomarker genes in WAT, some of which might play rolesin macrophage aggregation. Increased knowledge of themechanisms by which these inflammatory processes occurin scWAT and cause associated comorbidities is required.

ACKNOWLEDGMENTS

This work was supported by Institut National de la Santeet de la Recherche Medicale (INSERM) Avenir contract,the Direction de la Recherche Clinique/Assistance Pub-lique-Hopitaux de Paris, the Programme Hospitalier deRecherche Clinique (AOR02076 and clinical research con-tract no. 97123), and the Alfediam Association (clinicalresearch contract 2002/2003). R.C. and S.T. are funded byINSERM, ADR Paris 6, Saint-Antoine, Paris, and ConseilRegional de l’Ile-de-France. R.C. received a grant from theAssociation Francaise d’Etude et de Recherche sur l’Obesite/Roche (France). Funding of the EA3502 team was providedby the Servier Research Institute and the Benjamin DelessertInstitute, Paris 6 University, Paris, France. P.-A.S. receivedgrants from Swegene and AFA.

The authors thank Dr. Patrick Levy, Dr. Philippe Sellam,and Dr. Marianne Cerceau (Surgery Department, AP/HP,Hopital Hotel Dieu, Paris, France) for performance ofsurgery biopsies. We also thank Prof. Thierry Molina andDr. Mohib Morcos for support at the Jacques-DelarueDepartment of Anatomio-Pathology (Hopital Hotel Dieu,Paris, France) and Audrey Sicard, Annie Le Gall, Made-leine Gouillon, and Genevieve Bonhomme for their helpfultechnical assistance. We thank Dr. Michele Guerre-Millo,

FIG. 4. Changes in expression of macro-phage chemoattractant genes and hypox-ia-inducible gene in obesity and aftersurgery-induced weight loss. RTqPCR ofMCP-1 (A), CSF-3 (B), HIF-1� (C), andPLAUR (D) genes in scWAT specimens oflean control (women, n � 7; C, �) andmorbidly obese (women, n � 11; OB)subjects before (T0, f) and 3 monthsafter (3M, o) weight loss surgery.RTqPCR values are expressed in arbi-trary units after 18S mRNA normaliza-tion. Wilcoxon nonparametric paired test(OB and T0 vs. 3M) and Student’s t testwith unequal variance (OB vs. C) wereused. ***P < 0.001, **P < 0.01, *P < 0.05.



Dr. Daniele Lacasa, and Dr. Cecile Lubrano for criticalreading of this manuscript.

REFERENCES

1. Cottam DR, Mattar SG, Barinas-Mitchell E, Eid G, Kuller L, Kelley DE,Schauer PR: The chronic inflammatory hypothesis for the morbidityassociated with morbid obesity: implications and effects of weight loss.Obes Surg 14:589–600, 2004

2. Hotamisligil GS, Shargill NS, Spiegelman BM: Adipose expression of tumornecrosis factor-alpha: direct role in obesity-linked insulin resistance.Science 259:87–91, 1993

3. Fried SK, Bunkin DA, Greenberg AS: Omental and subcutaneous adiposetissues of obese subjects release interleukin-6: depot difference andregulation by glucocorticoid. J Clin Endocrinol Metab 83:847–850, 1998

4. Samad F, Yamamoto K, Pandey M, Loskutoff DJ: Elevated expression oftransforming growth factor-beta in adipose tissue from obese mice. Mol

Med 3:37–48, 19975. Sartipy P, Loskutoff DJ: Monocyte chemoattractant protein 1 in obesity

and insulin resistance. Proc Natl Acad Sci U S A 100:7265–7270, 20036. Samad F, Yamamoto K, Loskutoff DJ: Distribution and regulation of

plasminogen activator inhibitor-1 in murine adipose tissue in vivo: induc-tion by tumor necrosis factor-alpha and lipopolysaccharide. J Clin Invest

97:37–46, 19967. Lehrke M, Lazar MA: Inflamed about obesity. Nat Med 10:126–127, 20048. Trayhurn P, Wood IS: Adipokines: inflammation and the pleiotropic role of

white adipose tissue. Br J Nutr 92:347–355, 20049. Cancello R, Tounian A, Poitou C, Clement K: Adiposity signals, genetic and

body weight regulation in humans. Diabetes Metab 30:215–227, 200410. Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW

Jr: Obesity is associated with macrophage accumulation in adipose tissue.J Clin Invest 112:1796–1808, 2003

11. Xu H, Barnes GT, Yang Q, Tan G, Yang D, Chou CJ, Sole J, Nichols A, RossJS, Tartaglia LA, Chen H: Chronic inflammation in fat plays a crucial rolein the development of obesity-related insulin resistance. J Clin Invest

112:1821–1830, 200312. Curat CA, Miranville A, Sengenes C, Diehl M, Tonus C, Busse R, Bouloumie A:

From blood monocytes to adipose tissue-resident macrophages: induction ofdiapedesis by human mature adipocytes. Diabetes 53:1285–1292, 2004

13. Tchoukalova YD, Sarr MG, Jensen MD: Measuring committed preadipo-cytes in human adipose tissue from severely obese patients by usingadipocyte fatty acid binding protein. Am J Physiol Regul Integr Comp

Physiol 287:R1132–R1140, 200414. Clement K, Viguerie N, Poitou C, Carette C, Pelloux V, Curat CA, Sicard A,

Rome S, Benis A, Zucker JD, Vidal H, Laville M, Barsh GS, Basdevant A, StichV, Cancello R, Langin D: Weight loss regulates inflammation-related genes inwhite adipose tissue of obese subjects. FASEB J 18:1657–1669, 2004

15. Wellen KE, Hotamisligil GS: Obesity-induced inflammatory changes inadipose tissue. J Clin Invest 112:1785–1788, 2003

16. Esposito K, Pontillo A, Di Palo C, Giugliano G, Masella M, Marfella R,Giugliano D: Effect of weight loss and lifestyle changes on vascularinflammatory markers in obese women: a randomized trial. JAMA 289:1799–1804, 2003

17. Cummings DE, Overduin J, Foster-Schubert KE: Gastric bypass for obe-sity: mechanisms of weight loss and diabetes resolution. J Clin Endocrinol

Metab 89:2608–2615, 200418. Pinkney J, Kerrigan D: Current status of bariatric surgery in the treatment

of type 2 diabetes. Obes Rev 5:69–78, 200419. Suter M, Giusti V, Heraief E, Zysset F, Calmes JM: Laparoscopic Roux-en-Y

gastric bypass. Surg Endosc 17:603–609, 200320. Hsu SM, Raine L, Fanger H: Use of avidin-biotin-peroxidase complex (ABC) in

immunoperoxidase techniques: a comparison between ABC and unlabeledantibody (PAP) procedures. J Histochem Cytochem 29:577–580, 1981

21. Clement K, Viguerie N, Diehn M, Alizadeh A, Barbe P, Thalamas C, Storey JD,Brown PO, Barsh GS, Langin D: In vivo regulation of human skeletal musclegene expression by thyroid hormone. Genome Res 12:281–291, 2002

22. Tusher VG, Tibshirani R, Chu G: Significance analysis of microarraysapplied to the ionizing radiation response. Proc Natl Acad Sci U S A

98:5116–5121, 200123. Eisen MB, Spellman PT, Brown PO, Botstein D: Cluster analysis and

display of genome-wide expression patterns. Proc Natl Acad Sci U S A

95:14863–14868, 199824. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis

AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L,Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM,

Sherlock G: Gene ontology: tool for the unification of biology. The GeneOntology Consortium. Nat Genet 25:25–29, 2000

25. Ohlsson BG, Englund MC, Karlsson AL, Knutsen E, Erixon C, Skribeck H,Liu Y, Bondjers G, Wiklund O: Oxidized low density lipoprotein inhibitslipopolysaccharide-induced binding of nuclear factor-kappaB to DNA andthe subsequent expression of tumor necrosis factor-alpha and interleukin-1beta in macrophages. J Clin Invest 98:78–89, 1996

26. Su AI, Wiltshire T, Batalov S, Lapp H, Ching KA, Block D, Zhang J, SodenR, Hayakawa M, Kreiman G, Cooke MP, Walker JR, Hogenesch JB: A geneatlas of the mouse and human protein-encoding transcriptomes. Proc Natl

Acad Sci U S A 101:6062–6067, 200427. Nakajima T, Iikura M, Okayama Y, Matsumoto K, Uchiyama C, Shirakawa

T, Yang X, Adra CN, Hirai K, Saito H: Identification of granulocytesubtype-selective receptors and ion channels by using a high-densityoligonucleotide probe array. J Allergy Clin Immunol 113:528–535, 2004

28. Svensson PA, Hagg DA, Jernas M, Englund MC, Hulten LM, Ohlsson BG,Hulthe J, Wiklund O, Carlsson B, Fagerberg B, Carlsson LM: Identificationof genes predominantly expressed in human macrophages. Atherosclerosis

177:287–290, 200429. Muller WA: New mechanisms and pathways for monocyte recruitment. J

Exp Med 194:F47–F51, 200130. Murdoch C, Giannoudis A, Lewis CE: Mechanisms regulating the recruit-

ment of macrophages into hypoxic areas of tumors and other ischemictissues. Blood 104:2224–2234, 2004

31. Blasi F, Carmeliet P: uPAR: a versatile signalling orchestrator. Nat Rev Mol

Cell Biol 3:932–943, 200232. Bates SR, Murphy PL, Feng ZC, Kanazawa T, Getz GS: Very low density

lipoproteins promote triglyceride accumulation in macrophages. Arterio-

sclerosis 4:103–114, 198433. Linton MF, Fazio S: Macrophages, inflammation, and atherosclerosis. Int J

Obes Relat Metab Disord 27 (Suppl. 3):S35–S40, 200334. Li AC, Glass CK: The macrophage foam cell as a target for therapeutic

intervention. Nat Med 8:1235–1242, 200235. van Haarst JM, de Wit HJ, Drexhage HA, Hoogsteden HC: Distribution and

immunophenotype of mononuclear phagocytes and dendritic cells in thehuman lung. Am J Respir Cell Mol Biol 10:487–492, 1994

36. Terman A, Brunk UT: Lipofuscin. Int J Biochem Cell Biol 36:1400–1404, 200437. Brunk UT, Terman A: Lipofuscin: mechanisms of age-related accumulation

and influence on cell function. Free Radic Biol Med 33:611–619, 200238. Fain JN, Madan AK, Hiler ML, Cheema P, Bahouth SW: Comparison of the

release of adipokines by adipose tissue, adipose tissue matrix, andadipocytes from visceral and subcutaneous abdominal adipose tissues ofobese humans. Endocrinology 145:2273–2282, 2004

39. Mantovani A, Sozzani S, Locati M, Allavena P, Sica A: Macrophagepolarization: tumor-associated macrophages as a paradigm for polarizedM2 mononuclear phagocytes. Trends Immunol 23:549–555, 2002

40. Charriere G, Cousin B, Arnaud E, Andre M, Bacou F, Penicaud L, CasteillaL: Preadipocyte conversion to macrophage: evidence of plasticity. J Biol

Chem 278:9850–9855, 200341. Cousin B, Andre M, Casteilla L, Penicaud L: Altered macrophage-like

functions of preadipocytes in inflammation and genetic obesity. J Cell

Physiol 186:380–386, 200142. Cousin B, Munoz O, Andre M, Fontanilles AM, Dani C, Cousin JL,

Laharrague P, Casteilla L, Penicaud L: A role for preadipocytes asmacrophage-like cells. FASEB J 13:305–312, 1999

43. Lu B, Rutledge BJ, Gu L, Fiorillo J, Lukacs NW, Kunkel SL, North R, GerardC, Rollins BJ: Abnormalities in monocyte recruitment and cytokine ex-pression in monocyte chemoattractant protein 1-deficient mice. J Exp Med

187:601–608, 199844. Levine JA, Jensen MD, Eberhardt NL, O’Brien T: Adipocyte macrophage

colony-stimulating factor is a mediator of adipose tissue growth. J Clin

Invest 101:1557–1564, 199845. Kabon B, Nagele A, Reddy D, Eagon C, Fleshman JW, Sessler DI, Kurz A:

Obesity decreases perioperative tissue oxygenation. Anesthesiology 100:274–280, 2004

46. Koong AC, Denko NC, Hudson KM, Schindler C, Swiersz L, Koch C, EvansS, Ibrahim H, Le QT, Terris DJ, Giaccia AJ: Candidate genes for the hypoxictumor phenotype. Cancer Res 60:883–887, 2000

47. Semenza GL: Targeting HIF-1 for cancer therapy. Nat Rev Cancer 3:721–732,2003

48. Cavaillon JM: Cytokines and macrophages. Biomed Pharmacother 48:445–453, 1994

49. Wood GW, Hausmann E, Choudhuri R: Relative role of CSF-1, MCP-1/JE,and RANTES in macrophage recruitment during successful pregnancy. Mol

Reprod Dev 46:62–69, 1997 [discussion 69–70]



Original Article - Diabetes · and 3 months after bypass surgery. Immunomorphological changes of...

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